The goal of this research is to elucidate and characterize the mechanism of chromosome segregation during cell division in Caulobacter crescentus. Accumulating evidence suggests that many bacterial species, including Caulobacter crescentus, utilize DNA partitioning systems (Par systems) related to those found in plasmids to segregate portions of their chromosomes after DNA replication. Par systems are widespread and well studied in bacteria, yet the mechanism of DNA partitioning by these systems remains largely hypothetical. In this proposed research, both established and emerging technologies in cell biology will be utilized to examine the role and mechanism of the C. crescentus chromosomal Par system in the segregation of the C. crescentus chromosomal origin region. The structures and dynamic localization patterns of the two protein components of the Par system, the ATPase ParA and the DNA binding factor ParB, will be determined during chromosome segregation using timelapse and deconvolution fluorescence microscopy, and super- resolution PALM microscopy methods. The dynamics of ParA subunits within observed ParA structures will be examined using photobleaching/timelapse microscopy and single molecule tracking methods to directly test between predictions of various mechanistic models proposed for Par-induced segregation. Finally, the effect of mutating conserved ParA and ParB residues on DNA segregation will be determined to clarify the roles of the various molecular interactions in the chromosome segregation process. These experiments will lead to a complete mechanistic understanding of chromosome segregation by the C. crescentus Par system which will likely be applicable to both chromosomal and plasmid DNA segregation mechanisms in diverse bacterial species, Characterization of the DNA segregation mechanism in Caulobacter will undoubtedly elucidate novel targeting strategies for antibiotics that obstruct bacterial growth and inhibit the spread of drug-resistance between bacteria. PUBLIC HEALTH RELEVANCE: The goal of this research is to understand how bacteria partition DNA to their progeny during cell division. This fundamental process is essential for bacterial growth and infection. The elucidation and characterization of the mechanism of bacterial DNA segregation will reveal novel strategies and targets for antibiotics that prevent bacterial growth and lead to new cures for bacterial infections.